CN112725241A

CN112725241A - Pseudomonas chlororaphis and application thereof in prevention and treatment of leaf spot of phomopsis stolonifera

Info

Publication number: CN112725241A
Application number: CN202110175792.6A
Authority: CN
Inventors: 李磊; 李宝聚; 黄艺烁; 谢学文; 石延霞; 柴阿丽
Original assignee: Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
Current assignee: Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
Priority date: 2021-02-09
Filing date: 2021-02-09
Publication date: 2021-04-30
Anticipated expiration: 2041-02-09
Also published as: CN112725241B

Abstract

The invention discloses pseudomonas aeruginosa and application thereof in preventing and treating a leaf spot disease of phoma stolonifera. The Pseudomonas chlororaphis is Pseudomonas chlororaphis YS05 which is preserved in China general microbiological culture Collection center on year 2020, 07-08, with the preservation number of CGMCC No. 20323. The invention also discloses application of the pseudomonas chlororaphis YS05 in inhibiting pathogenic bacteria. In the bacteriostasis spectrum determination, the strain YS05 shows broad-spectrum resistance and has antagonistic effect on 9 pathogenic fungi and 3 pathogenic bacteria. In a test for testing the prevention effect of the greenhouse pot culture, the prevention effect of the strain YS05 on the leaf spot of the stemphylium botryoides is 61.27%. In the test for determining the prevention effect of the in vitro leaves of the photinia stolonifera on the tomatoes, the prevention effect of the strain YS05 on the gray leaf spot of the tomatoes is 71.52%. The strain YS05 has better application prospect.

Description

Pseudomonas chlororaphis and application thereof in prevention and treatment of leaf spot of phomopsis stolonifera

Technical Field

The invention belongs to the technical field of biology, and particularly relates to pseudomonas aeruginosa and application thereof in prevention and treatment of leaf spot of phoma stolonifera in tomatoes.

Background

The pathogenic bacteria of the tomato Stemphylium leaf spot disease are Stemphylium solani (Stemphylium solani Weber) and Stemphylium solani (Enjoji) Yamamoto), mainly damage tomato leaves and severely spread to stems and fruits. The disease spots are brown spots which are gradually enlarged to be round or nearly round, and are easy to perforate and break at the later stage of disease incidence, thereby causing serious loss. At present, the control measures of tomato diseases depend on chemical control for a long time, but the problems of chemical residue, environmental pollution, drug-resistant pathogenic strains and the like caused by the large-scale application of synthetic pesticides are increasingly highlighted. In recent years, biological control has become a research hotspot due to the advantages of safety, high efficiency, economy, environmental protection and the like.

Pseudomonas spp is widely distributed in plant rhizosphere soil, and a plurality of strains can effectively inhibit pathogens and promote the growth and yield increase of plants, so that the Pseudomonas spp is concerned by researchers in various countries and is a biocontrol bacterium and a rhizosphere growth-promoting bacterium which are researched and reported most at home and abroad. In more than 30 years, the research on the biological control of pseudomonas mainly focuses on the growth promotion of rhizosphere, and in recent years, some scholars successfully control the infectious diseases of various plants such as wheat, strawberries, pakchoi, tomatoes and the like by using pseudomonas, so that the application range of the pseudomonas is greatly expanded.

Disclosure of Invention

The invention aims to provide pseudomonas chlororaphis and application thereof in prevention and treatment of leaf spot of phomopsis stolonifera.

In order to achieve the above object, the present invention firstly provides Pseudomonas chlororaphis subsp.aurantiaca YS05 with a preservation number of CGMCC No. 20323.

The Pseudomonas chlororaphis subsp. aurantiaca YS05 provided by the invention has been preserved in China general microbiological culture Collection center (CGMCC for short; address: No. 3 of West Lu 1 of Beijing, Inward region, China academy of sciences, Microbiol research institute; zip code: 100101) in 2020 and the preservation number is CGMCC No.20323 in 2020 and 08 months.

In order to achieve the above object, the present invention further provides a suspension or a culture solution or a fermentation product or a volatile substance of the above Pseudomonas chlororaphis subsp.

In order to achieve the above object, the present invention further provides use of a bacterial suspension or culture solution or fermentation broth or fermentation product or volatile substance of the above Pseudomonas chlororaphis subsp.

The invention also provides application of the bacterial suspension or culture solution or fermentation liquor or fermentation product or volatile substance of the Pseudomonas chlororaphis subsp.

The invention also provides application of the bacterial suspension, the culture solution, the fermentation liquor, the fermentation product, the volatile substance or the microbial inoculum containing the bacterial suspension, the culture solution, the fermentation liquor or the volatile substance of the Pseudomonas chlororaphis subsp.

In order to achieve the above object, the present invention also provides a product, the active ingredient of which is a bacterial suspension or culture solution or fermentation product or volatile substance of the above Pseudomonas chlororaphis subsp.

The product has any one of the following functions of a1) -a 3):

a1) inhibiting pathogenic bacteria;

a2) preventing and treating plant diseases caused by pathogenic bacteria;

a3) preventing and treating the leaf spot of the botrytis cinerea.

To achieve the above object, the present invention finally provides any one of the following methods b1) -b 3):

b1) a method of suppressing a pathogenic bacteria comprising the steps of: treating pathogenic bacteria with the above bacterial suspension or culture solution or fermentation broth or fermentation product or volatile substance of Pseudomonas chlororaphis subsp.

b2) A method of controlling plant diseases caused by pathogenic bacteria, comprising the steps of: treating the plant with a bacterial suspension or culture solution or fermentation broth or fermentation product or volatile substance of the above Pseudomonas chlororaphis subsp.

b3) A method for preventing and treating a leaf spot disease of tomato stemphylium stolonifera comprises the following steps: treating tomato with the above bacterial suspension or culture solution or fermentation product or volatile substance of Pseudomonas chlororaphis subsp.

Any of the above-mentioned pathogenic bacteria are plant pathogenic bacteria. The plant pathogenic bacteria can be pathogenic fungi or pathogenic bacteria.

The pathogenic fungi may specifically include anthrax (Colletotrichum spp.), Botrytis cinerea (Botrytis cinerea), corynebacterium polystachyum (Corynespora cassicola), Phytophthora capsici (Phytophthora capsici), Rhizoctonia solani (Rhizoctonia solani), Fusarium solani (Fusarium solani), Botrytis cinerea (stemphytopersicum lycopersici (Enjoji) Yamamoto), Alternaria solani (Alternaria solani), and Ascochyta citrullina (ascochyrlina).

The pathogenic bacteria may specifically include Corynebacterium michiganensis subsp, Agrobacterium vitis, Pseudomonas syringae var.

Any one of the plant diseases can be specifically a tomato stemphylium stolonifera leaf spot.

Any of the above plants may specifically be tomato.

The concentration of Pseudomonas chlororaphis subsp.aurantiaca YS05 in any one of the above bacterial suspensions, culture solutions, fermentation products, or microbial agents can be 1 × 10⁷cfu/mL-1×10⁹cfu/mL, specifically 1X 10⁸cfu/mL。

In a specific embodiment, the specific preparation method of the bacterial suspension or culture solution or fermentation product or microbial inoculum can comprise the following steps: inoculating Pseudomonas chlororaphis subsp. aurantiaca YS05 in KB liquid culture medium, shake culturing at 28 deg.C and 180r/min for 24 hr, and adjusting the bacterial suspension concentration to OD₆₀₀＝0.8。

In another embodiment, the specific preparation method of the bacterial suspension or culture solution or fermentation product or microbial inoculum can comprise the following steps: inoculating Pseudomonas chlororaphis subsp. aurantiaca YS05 in KB liquid culture medium, shake culturing at 28 deg.C and 180r/min for 24 hr, and adjusting the bacterial suspension concentration to OD₆₀₀0.8, and then diluted to 100 volumes with distilled water.

The invention separates a strain YS05 which has obvious antagonism to tomato stemphylium stolonifera from the rhizosphere soil of crops. The strain YS05 is preliminarily identified as the Pseudomonas chlororaphis subsp. In the bacteriostasis spectrum determination test, the strain YS05 shows broad-spectrum resistance and has antagonistic effect on 9 pathogenic fungi and 3 pathogenic bacteria. In a test for testing the prevention effect of the greenhouse pot culture, the prevention effect of the strain YS05 on the leaf spot of the stemphylium botryoides is 61.27%. In the test for determining the prevention effect of the in vitro leaves of the tomato stemphylium stolonifera, the prevention effect of the strain YS05 on the leaf spot of the tomato stemphylium stolonifera is 71.52%. The strain YS05 has good application prospect, and provides certain theoretical basis and technical support for further development and utilization.

Drawings

FIG. 1 shows the determination of antagonistic effect of 23 Pseudomonas strains on Pythium ultimum.

FIG. 2 shows the morphological characteristics of the strain YS 05. A: observing through transmission observation; b: observing by a scanning electron microscope; c: observing the front morphology of a culture medium of the strain YS05 KB; d: strain YS05KB medium reverse morphology observation.

FIG. 3 shows that the strain YS05 constructs a multigenic phylogenetic tree based on 16S rDNA, gyrB, rpoB and rpoD gene sequences.

FIG. 4 shows the bacterial inhibition spectrum of strain YS 05. A: the pathogenic bacterium Colletotrichum spp of pepper anthracnose; b: the pathogen Botrytis cinerea (Botrytis cinerea) of tomato gray mold; c: the pathogenic bacterium, corynebacterium polyspora (Corynespora cassiicola), of target spot disease in cucumber; d: phytophthora capsici (Phytophthora capsicii), a pathogenic bacterium of Phytophthora capsici; e: rhizoctonia solani (Rhizoctonia solani) which is a pathogenic bacterium of stem rot of Chinese cabbage; f: fusarium solani (Fusarinm solani), a pathogenic bacterium of melon root rot; g: pathogenic bacteria of tomato stemphylium heterosporum stolonifera (stemphyllumlycopersici (enjoji) Yamamoto); h: the pathogenic bacterium of potato early blight Alternaria solani (Alternaria solani); i: the pathogenic bacterium of gummy stem blight is exocarpium citrulli citrullina; j: the pathogenic bacteria of tomato canker, corynebacterium michiganensis subsp. (corynebacterium michiganensis); k: the pathogenic bacteria of grape crown gall disease, Agrobacterium vitis (Agrobacterium vitis); l: pathogenic bacteria of bacterial spot disease in tomato, Pseudomonas syringae tomato pathogenic variety (Pseudomonas syringaepv. tomato).

FIG. 5 shows the determination of the biocontrol factor of strain YS 05. A: a protease; b: HCN; c: antibiotic resistance;

FIG. 6 shows the PCR product gel of the common antibiotic gene of strain YS 05. M: DNA maker BM 5000; 1: negative control; 2: pseudomonas fluorescens 2P 24; 3: YS 05.

FIG. 7 shows the measurement of the volatile substance inhibitory effect of the strain YS 05.

FIG. 8 shows the determination of the prevention effect of the strain YS05 on the bonsai of the tomato stemphylium stolonifera. A: 100 times of bacterial suspension of the strain YS 05; b: 100 times of bacterial suspension of the strain YS 21; c: iprodione 800 times liquid; d: distilled water control; e: healthy control.

FIG. 9 shows the determination of the prevention effect of the strain YS05 and 6 medicaments on the in vitro leaves of the photinia stolonifera. A: bacillus cereus wettable powder; b: a Bacillus licheniformis aqueous agent; c: 46% copper hydroxide water dispersible granules; d: 33.5% oxine-copper suspension; e: wettable powder of 10% polyoxin; f: 50% iprodione wettable powder; g: bacterial suspension of strain YS 05; h: blank control (distilled water).

Deposit description

The name of Chinese: pseudomonas chlororaphis subspecies aurantiaca

Latin name: aurantiaca of Pseudomonas chlororaphis subsp

The strain number is as follows: YS05

The preservation organization: china general microbiological culture Collection center

The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)

Address: xilu No. 1 Hospital No. 3 of Beijing market facing Yang district

The preservation date is as follows: year 2020, month 07 and 08

Registration number of the preservation center: CGMCC No.20323

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The medium in the following examples is naturally pH unless otherwise specified. Unless otherwise stated, the quantitative tests in the following examples were performed in triplicate, and the results were averaged.

The tomato variety to be tested in the following examples is 'Zhongza 201' purchased from vegetable and flower institute of Chinese academy of agricultural sciences.

The media in the following examples are as follows:

KB solid medium (for isolation and purification of pseudomonas): peptone 20.0g, glycerol 10.0mL, MgSO₄·7H₂O 1.5g，K₂HPO₄1.5g, agar 20g, and distilled water 1000 mL.

KB liquid medium (for shake culture and propagation of pseudomonas): peptone 20.0g, glycerol 10.0mL, MgSO₄·7H₂O 1.5g，K₂HPO₄1.5g, 1000mL of distilled water.

WA medium (for determination of bacterial inhibition profile): agar 5g and distilled water 1000mL, and the mixture was dispensed into 5mL test tubes.

PDA medium (for fungal antagonism test): 200.0g of potato, 20.0g of glucose, 20.0g of agar and 1000mL of distilled water

CMC medium (for cellulase assay): MgSO (MgSO)₄ 0.1g、(NH₄)₂SO₄1.0g, 100.0mL of 10 XPhospate Buffer, 5.0g of yeast powder, 2.0mL of glycerol, 1.0g of CMC, 12.0g of Bacto Agar and 1000mL of distilled water.

Skim milk medium (for protease assay): 10.0g of peptone, 3.0g of beef powder, 5.0g of NaCl, 1000mL of water and 100mL of skimmed milk, and the pH value is 7.0.

Congo red solution: 0.2% (W/V), 2g of Congo red powder and 1000mL of distilled water, and a small amount of alcohol can be added to help the uniform dissolution.

NaCl solution: 58.5g of 1M NaCl, 1000mL of distilled water.

Example 1 isolation, screening, identification and preservation of Strain YS05

Separation and purification of strain YS05

Taking rhizosphere soil samples of open field crops from Harbin city, weighing 10g of each soil sample, adding into 90mL of sterilized water, and placing in a constant temperature shaking table at 28 ℃ for shaking culture for 30 min. According to 10^-1-10^-7Performing gradient dilution, and taking dilution gradient 10^-5、10^-6、10^-7The soil sample is plated and cultured in a constant temperature incubator at 28 ℃ for 24 hours. After the coated plate grows out single colonies, selecting the single colonies with different forms by using a sterilizing toothpick, streaking the single colonies on a KB solid plate, purifying the single colonies, culturing the single colonies in a constant-temperature incubator at 28 ℃ until the single colonies with the consistent forms grow out, and storing the single colonies in a refrigerator at 4 ℃ for later use. And finally, 236 bacteria are obtained by co-separation from rhizosphere soil of the Harbin open field crop.

Second, screening of Strain YS05

And (3) determining the inhibition rate of the separated and purified bacteria on the stemphylium botryoides of the tomatoes by adopting a plate confronting method. The method comprises the following specific steps: inoculating tomato stemphylium pratense cake with diameter of 5mm at the center of PDA plate, and respectively dripping 5 μ L of OD at 4 points 3cm away from the center of the plate by cross method₆₀₀The bacterial liquid to be tested is cultured at 28 ℃ by taking the non-inoculated antagonistic strain as a control, and the investigation is carried out when the plate of the non-inoculated antagonistic strain grows to the edge of the plate. Each treatment was repeated 3 times. The control colony diameter and the treated colony diameter of the target bacteria were measured and expressed as the inhibition rate. Inhibition (%) - (control colony diameter-treated colony diameter)/control colony diameter]×100。

The antagonistic bacteria having an inhibitory effect on Phoma stolonifer of tomato were obtained in 23 strains by plate confrontation, and the strains were numbered YS01-YS 23. The antagonistic effect of 23 strains of antagonistic bacteria on ustilaginoidea lycopersicum is shown in table 1 and fig. 1, and the results show that: the 23 antagonistic bacteria have a good inhibition effect on the tomato stemphylium stolonifera, and the growth of hyphae is inhibited, wherein the strain YS05 has the most obvious effect, and the inhibition rate reaches 68.91%.

TABLE 1 determination of antagonistic Effect of 23 strains of Pseudomonas on Podospora solani

Original numbering	Numbering	Preliminary identification results	Inhibition ratio%
				Scallion-1	YS01	Pseudomonas chlororaphis	44.83±2.64hij
Scallion-31	YS02	Pseudomonas fluorescens	0.00±0.00l
				Potato-14	YS03	Pseudomonas fluorescens	43.32±1.93ij
Sunflower-3	YS04	Pseudomonas chlororaphis	58.34±3.77bcd
				YS05	YS05	Pseudomonas chlororaphis	68.91±7.29a
Sunflower-4	YS06	Pseudomonas chlororaphis	46.17±1.94ghij
				Sunflower-9	YS07	Pseudomonas chlororaphis	48.36±3.18ghi
Sunflower-16	YS08	Pseudomonas fluorescens	41.62±2.22j
				Sunflower-18	YS09	Pseudomonas fluorescens	45.37±0.68hij
Sunflower-20	YS10	Korean Pseudomonas	35.04±1.45k
				Sunflower-22	YS11	Pseudomonas chlororaphis	50.36±1.59fgh
Sunflower-32	YS12	Pseudomonas fluorescens	41.74±1.19j
				XRK01	YS13	Pseudomonas chlororaphis	52.05±2.00efg
XRK03	YS14	Pseudomonas chlororaphis	56.34±2.62cde
				Flax-7	YS15	Korean Pseudomonas	34.36±4.36k
Flax-13	YS16	Pseudomonas fluorescens	54.65±7.32def
				Flax-16	YS17	Pseudomonas chlororaphis	49.90±1.30fgh
Corn-5	YS18	Pseudomonas chlororaphis	63.06±1.27b
				Corn-6	YS19	Korean Pseudomonas	33.77±6.70k
Corn-9	YS20	Pseudomonas chlororaphis	48.15±1.42ghi
				Corn-22	YS21	Pseudomonas chlororaphis	60.93±1.01bc
Corn-24	YS22	Pseudomonas fluorescens	44.32±0.82hij
				YM28	YS23	Pseudomonas chlororaphis	54.33±1.13def

Third, identification of Strain YS05

1. Morphological identification

Morphological feature observation was performed on the strain YS 05.

The results are shown in FIG. 2 and show that: pseudomonas chlororaphis subspecies aurantiaca YS05 appears milky white on the front side and orange on the back side in a KB plate, and the strain is rod-shaped and single flagellum is polar under an electron microscope and has the size of (0.8-1.1) × (1.2-2.7) mu m.

2. Physiological and biochemical identification

The following physiological and biochemical tests were carried out on the strain YS05 with reference to methods in the book of identification of common bacteria systems (Dongxu bead, Chuiamiao English, 2001. Manual of identification of common bacteria systems, Beijing: scientific Press) and in the Manual of identification of Bergey's bacteria (Bukannan R E.1984. Bogey's Manual of identification of bacteria, Beijing: scientific Press): gram test, growth temperature test, salt tolerance test, motility test, catalase test, V-P test, starch hydrolysis test, glucose oxidation fermentation test, gelatin liquefaction test, nitrate reduction test and the like.

The results are shown in table 2 and show that: the test results of the strain YS05, such as salt tolerance, catalase, V-P reaction, starch hydrolysis, nitrate reduction and the like, are consistent with the reaction characteristics of the pseudomonas chlororaphis HL5-4 and the pseudomonas chlororaphis Pa40, and the strain is primarily determined to be the pseudomonas chlororaphis.

TABLE 2 identification of physiological and biochemical characteristics

Note: + is positive; -is indicated as negative.

3. Biolog assay

And selecting a YS05 single colony, inoculating the single colony to a KB culture medium test tube inclined plane, culturing at the constant temperature of 28 ℃ for 24h, and determining the utilization of the unique carbon source by using a BIOLOG GEN III kit (operated according to the kit specification) by the China agricultural microbial strain collection center.

The use of the unique carbon source on the BIOLOG GENIII reagent strip by strain YS05 is shown in Table 3.

TABLE 3 determination of the sole carbon Source utilization of Strain YS05 Using the BIOLOG GENIII reagent strips

Note: positive; -, negative; w, weak positive.

4. Molecular identification

After the genomic DNA of the strain YS05 is extracted in a small quantity by using the TIANGEN bacterial genomic DNA extraction kit, 16S rDNA, gyrB, rpoB and rpoD genes are selected for amplification and sequencing. 16S rDNA gene amplification primer-27F: 5'-AGAGTTTGATCCTGGCTCAG-3', respectively; 1492R: 5'-AAGGAGGTGATCCAGCCGCA-3' are provided. gyrB gene amplification primer-gyrB 1F: 5'-GACAAGCTGGTGTCTTCCGA-3', respectively; gyrB 1R: 5'-CATCTCGCCCAGACCTTTGT-3' are provided. rpoB gene amplification primer-rpoB 1F: 5'-GTCCGACTTCACTGTGAGCA-3', respectively; rpoB 1R: 5'-GTGAAAAAGCCAGCGACGTT-3' are provided. rpoD gene amplification primer-rpoD 1F: 5'-CACGGTTGAGCACATCCTCT-3', respectively; rpoD 1R: 5'-GGAGAGTACTTCGCGAGTCG-3' are provided.

The PCR amplification system is 25 mu L, and comprises 0.5 mu L of each of the upstream primer and the downstream primer and 1 mu L of template DNA; 12.5 mu L of T-Taq Mix; ddH₂O 10.5μL。

PCR reaction procedure: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 45s for 34 cycles; extension at 72 ℃ for 10 min.

The resulting PCR product was sequenced by Bomader Biometrics. The sequencing result shows that: the 16S rDNA, gyrB, rpoB and rpoD gene sequences of the strain YS05 are respectively shown as sequences 1-4 in the sequence table. And (3) downloading the rest sequences by the NCBI website, connecting and comparing the 16S rDNA, gyrB, rpoB and rpoD gene sequences of different strains by using MEGA 7.0, Sequence Matrix and Seaview4 in Sequence, constructing a phylogenetic tree by adopting a maximum likelihood method, and analyzing the classification status.

Based on rDNA, gyrB, rpoB and rpoD gene sequences of strains YS 0516S, a YS05 polygene phylogenetic tree is constructed, and the result is shown in figure 3, and the result shows that: the strain YS05 and P.chlorophyllis subsp.aurantiaca LMG 21630 of Pseudomonas aeruginosa subsp.aurantiaca clustered in one branch.

Combining the results of morphology, physiological and biochemical characteristics, Biolog detection and molecular identification, the strain YS05 was determined to belong to the species Pseudomonas chlororaphis subsp.

Fourth, preservation of Strain YS05

Pseudomonas chlororaphis subsp. aurantiaca YS05 has been deposited in China general microbiological culture Collection center (CGMCC; address: Beijing city, Chaoyang district, West Lu No. 1 Hospital No. 3, institute of microbiology, China academy of sciences; postal code: 100101) at 2020 and 08 th year in 2020, with the deposition number of CGMCC No. 20323.

Example 2 determination of the bacterial inhibition Spectrum of Strain YS05

Common 9 vegetable pathogenic fungi and 3 pathogenic bacteria are selected to determine the bacterial inhibition spectrum of the strain YS 05.

The 9 vegetable pathogenic fungi and 3 pathogenic bacteria are shown in table 4, and the specific origins are as follows:

anthrax (Colletotrichum spp.) has been reported in "ocean, sauvignon, fount, li bao-polyp. compatible pathogenic bacteria induce disease resistance mechanism of cucumber tissue structure. plant protection article 2008 (01): 37-42 ", publicly available from the vegetable and flower institute of Chinese academy of agricultural sciences to repeat the experiments of the present application, and not for other uses.

Botrytis cinerea has been described in the literature "resistance of Botrytis cinerea to different types of fungicides" Botrytis cinerea "in Cabernet, Tangming, Genzhi, Schering, Chailali, Li Bao. 60-65, publicly available from the vegetable and flower institute of Chinese academy of agricultural sciences to repeat the experiments of the present application, and not for other uses.

Corynebacterium polystachyum (Corynespora cassicola) has been described in the literature "homosala, lipamopsis, eucrypti, schwerwinia". pathogenic differentiation of corynebacterium polystachyum in cucumber, tomato and eggplant hosts. 465- & 470 ", the public can obtain from the vegetable flower research institute of Chinese academy of agricultural sciences to repeat the experiments of the present application and cannot be used for other purposes.

Phytophthora capsici (Phytophthora capsicii) has been reported in the literature "Zhuhui, Wang Happy, Li Bao Ju, Cabernet Sauvignon. identification of pathogen of root rot type Phytophthora blight and screening of control agent. plant protection journal 2007, (4): 373-' 378. discloses that the public is available from the vegetable and flower institute of Chinese academy of agricultural sciences to repeat the experiments of the present application and not be used for other purposes.

Rhizoctonia solani (Rhizoctonia solani) has been reported in the literature "antagonism of trichoderma viride against Rhizoctonia solani and fusarium oxysporum f.cucumeri, sorrel, grandsond, sauvigna, tsukushinensis., (iii) chinese vegetable 2008, (6): 9-12 ", publicly available from the vegetable and flower institute of Chinese academy of agricultural sciences to repeat the experiments of the present application, and not for other uses.

Fusarium solani (Fusarium solani) has been described in the literature "cardia petrel, Shinya, Xiechuan, Chailali, Li Bao-poly. calcium cyanamide soil disinfection control of cucumber root rot and soil pathogens. horticultural journal 2016, 43 (11): 2173 and 2181, the public is available from the vegetable and flower institute of Chinese academy of agricultural sciences to repeat the experiments of this application and not for other uses.

Staphylium lycopersicum (Enjoji) Yamamoto has been documented in "plum blossoms, perigory aromas, li jinju, scholaria, diagnosis and control of staphylium leaf spot (gray leaf spot) of tomato, chinese vegetables 2010, (23): 24-26 ", publicly available from the vegetable and flower institute of the Chinese academy of agricultural sciences to repeat the experiments of the present application and not for other uses.

Alternaria solani (Alternaria solani) has been described in the literature "chairali, sauvignon, academy, patiguli, libanopsis john.processed tomato early blight pathogen identification. north china agricultural press 2015, 30 (supplement): 316-.

The species Ascochyta citrullina is disclosed in the literature "SHI Yanxia, MENG Shanshan shan, XIE Xuewen, CHAI Ali, LI Baoju. Dry Heat Treatment Reduces of the Occurrence of Cladosporium cumerium, Ascochyta citrullina, and Colpolytrichum orbiculare on the Surface and interface of cell section Plant Horticultural Plant Journal 2016,2(01): 35-40", publicly available from the vegetable and flower institute of the national academy of agricultural sciences for the purpose of repeating the experiments of the present application and not for other uses.

Corynebacterium michiganensis subsp. michiganensis has been disclosed in "li huan, huixia, schwann, li bao, the occurrence rule and prevention and treatment technique of tomato canker" chinese vegetables 2011, 23:24-27 ", publicly available from vegetable and flower research institute of chinese academy of agricultural sciences, to repeat the application experiment, and is not applicable for other uses.

Agrobacterium vitis (Agrobacterium vitis) has been described in "zhao yi banyan, li yu yi, xie weng, xianya, bairali, sun guangye, li bao pi.beleisi bacillus ZF2 has control effect on polyspora corynomycosis. 217 + 225 ", publicly available from the vegetable and flower institute of Chinese academy of agricultural sciences to repeat the experiments of the present application and not for other uses.

Pseudomonas syringae tomato pathogenic variety (Pseudomonas syringae pv. tomato) has been described in the literature "chaarali, patiguli, gournet, cabbages, schlieren, schimmer, plum blossom. establishment and application of real-time fluorescent quantitative PCR detection method of tomato bacterial spot pathogen. 182. "to be published, the public can obtain from the vegetable flower institute of the Chinese academy of agricultural sciences to repeat the experiments of the present application, and cannot be used for other purposes.

The inhibitory effect of the strain YS05 on pathogenic bacteria was determined by plate confrontation (fungi) and double layer culture (bacteria), respectively. Assay method references "Stonier t.1960.agrobacterium tumefaciens conn.ii.process of an antigenic substance. journal of Bacteriology, 79 (6): 889 ", the method comprises the following steps: strain YS05 glycerol was taken from-80 deg.C refrigeratorThe tube was aspirated 1mL of the solution, added to a 250mL conical flask containing 100mL of KB medium, shaken at 28 ℃ and 180rmp/min for 24 hours, and then OD was measured₆₀₀Values and adjustment of OD Using blank KB liquid Medium₆₀₀The bacterial suspension was inoculated in a KB plate center at 5. mu.L, incubated at 28 ℃ for 24h, and then inactivated by fumigation with chloroform, as a lower auxin layer, for use at 0.8. 5mL of WA medium containing pathogenic bacteria WAs used as the upper layer. After culturing at 28 ℃ for 48h, the diameter of the inhibition zone is measured, the inhibition rate is calculated, and each treatment is repeated for 3 times. Inhibition (%) - (control colony diameter-treated colony diameter)/control colony diameter]×100。

The results are shown in table 4 and fig. 4, and show that: the strain YS05 can effectively inhibit 7 fungi including Botrytis cinerea, Cladosporium polystachyum, Phytophthora capsici, Rhizoctonia solani, anthrax, Alternaria solani and Podospora solani, the inhibition rate is more than 50%, and the inhibition effect on Fusarium solani and Sepiella citrulli is relatively weak, namely 21.4% and 7.73%. The strain YS05 has good inhibitory effect on pathogenic bacteria, the diameter of inhibiting the epiphora pathogenic variety of the clove pseudomonas is 4.37cm, and the diameter of inhibiting the rhizopus is 4.85 cm.

TABLE 4 bacteriostatic effect of the strain YS05 on pathogenic bacteria

Note: data are mean ± sem, with different small letters indicating significant differences at the 0.05 level. The + inhibition zone is larger than 0.5cm, the + inhibition zone is larger than 1cm, the + inhibition zone is larger than 2cm, and the-inhibition effect is not achieved.

Example 3 determination of biocontrol factor for Strain YS05

1. Detection of cellulase

Inoculating 5 μ L OD in the center of CMC medium₆₀₀Culturing the strain YS05 bacterial suspension at 28 ℃ for 24h, adding 3mL Congo red dye, dyeing for 30min, pouring off the dye, adding 5mL of 1mol/L NaCl solution, decoloring for 15min, and observing a digestion loop. Each treatment was repeated 3 times.

2. Detection of proteases

Inoculating 5 μ L of OD in the center of skim milk medium₆₀₀The bacterial suspension of 0.8 strain YS05 was cultured at 28 ℃ for 24 hours, and then the digestion loop was observed. Each treatment was repeated 3 times.

3. Detection of hydrocyanic acid HCN

Cutting Whatman filter paper into 5mm × 25mm paper strips; 25mg of copper (II) ethyl acetate and 25mg of 4-4' -methyenebis- (N, N-dimethyllaniline) were weighed out separately and dissolved in 10mL of chloroform, and after sterilization with tweezers, strips of Whatman filter paper were soaked completely in the above solution, air-dried, and stored in 9cm glass petri dishes for use.

The results are shown in FIG. 5 and show that: the strain YS05 is produced in a transparent circle in a skimmed milk culture medium; inoculation of strain YS05 the test strips turned blue. The fact that the metabolite of the strain YS05 contains protease and hydrocyanic acid HCN has certain biocontrol potential is shown.

4. Antibiotic resistance assay

1) The resistance of strain YS05 to 10 antibiotics (Table 5) was determined. The method comprises the following specific steps: 3mL of liquid medium containing antibiotic KB were added to a 15mL sterile tube and inoculated with 1% OD₆₀₀A suspension of YS05 strain was shaken in sterile tubes for 24h and the strains were observed for antibiotic resistance and repeated 3 times for each treatment, 0.8.

TABLE 5 antibiotic formulation

Classes of antibiotics	Concentration of use	Solvent(s)
			Ampicillin ([ Amp ]]Ampicillin)	100mg/mL	Water (W)
Carbenicillin ([ Car)]Carbenicillin)	50mg/mL	Water (W)
			Kanamycin ([ Kan)]Kanamycin)	50mg/mL	Water (W)
Chloramphenicol ([ Cm)]Chloramphenicol)	25mg/mL	Anhydrous ethanol
			Streptomycin ([ Str)]Streptomycin)	50mg/mL	Water (W)
Tetracycline ([ Tet)]Tetracyyline)	10mg/mL	Anhydrous ethanol
			Gentamicin ([ Gm)]Gentamicin)	50mg/mL	Water (W)
Rifampicin ([ Rif)]Rifampicin)	50mg/mL	Dimethyl sulfoxide
			Vancomycin ([ Van ]]Vancomycin)	50mg/mL	Water (W)
Spectinomycin or spectinomycin ([ Spec ]]Spectinomycin)	50mg/mL	Water (W)

The results show that: the strain YS05 is tolerant to antibiotics such as ampicillin, chloramphenicol, vancomycin, carbenicillin, streptomycin, spectinomycin and the like, and is not tolerant to four antibiotics such as kanamycin, rifampin, tetracycline, gentamicin and the like.

2) Amplification of common antibiotic gene of strain YS05

After extracting genomic DNA of the strain YS05, the genomic DNA was PCR-amplified using each primer set shown in Table 6.

TABLE 6 antibiotics and corresponding Gene names

And (3) PCR reaction system: 1 uL of upstream primer, 1 uL of downstream primer, 1 uL of DNA template, 25 uL of T-Taq Mix, ddH₂O 22μL。

PCR reaction procedure: pre-denaturation at 94 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 90s for 34 cycles; extending for 10min at 72 ℃; storing at 12 deg.C. A temperature gradient is set. And (3) performing PCR reaction by taking pseudomonas fluorescens 2P24 as a control, taking 5 mu L of PCR product, performing 1% agarose gel electrophoresis verification, and sequencing the product to determine the similarity of the biosynthesis gene sequence of the antibiotic generated by the screened biocontrol bacteria.

The gel appearance result of the PCR product of the common antibiotic gene of the strain YS05 is shown in FIG. 6, and the PCR amplification result shows that: the strain YS05 contains genes for coding synthetic Phenazine Phenazine and nitropyrrolidin Pyrrolnitrin, and 2,4-DAPG, pyoluteorin and 2-hydroxyphenylazine genes are not amplified.

Example 4 determination of bacteriostatic Activity of the volatile substance of Strain YS05

The biocontrol strain YS05 and pathogenic bacteria (the pathogenic bacteria are shown in Table 7) are respectively cultured by using a two-section dish, and the pathogenic fungus inhibition effect of the volatile substance of the strain YS05 is measured. The method comprises the following specific steps: the strain YS05 was taken out from a refrigerator at-80 ℃ and activated using a 15mL shake tube containing 3mL KB of liquid medium, and then shaken at 28 ℃ and 180rpm/min for 24 hours to obtain a seed solution. Transferring the seed solution into a 250mL triangular flask containing 200mL KB of liquid culture medium, shaking for 24h, and determining OD₆₀₀Is 0.8. Adding a PDA culture medium into the two halves of dishes, inoculating the strain YS05 by adopting a line drawing method, adding a 5mm pathogenic fungus cake into the corresponding side, placing the two halves of dishes in an incubator at 28 ℃ for inverted culture, taking no biocontrol bacteria as a control, observing that the pathogenic fungi of a blank control grow to the edge of the plate for investigation, and repeating the treatment for 3 times.

The results are shown in table 7 and fig. 7, and show that: the volatile matter of the strain YS05 has the inhibition rate of more than 40 percent on rhizoctonia solani of vegetables and fusarium cucumerinum and the inhibition rate of about 10 percent on fusarium solani of melons.

TABLE 7 measurement of the inhibitory Effect of the volatile substance by Strain YS05

Example 5 determination of Effect of Strain YS05 on controlling leaf spot of tomato stemphylium stolonifera

And (3) carrying out the prevention effect determination of the leaf spot of the stemphylium botryoides by adopting a spray inoculation method.

The specific method comprises the following steps: when the potted tomato grows to 5 true leaves, the following treatments are respectively carried out:

treatment group 1: OD mixing with sterile Water₆₀₀Diluting the suspension of 0.8 strain YS05 by 100 times, spraying on the plants, and spraying 5 strains of the liquid medicine on the leaves without dripping water0mL of treatment solution (OD)₆₀₀Bacterial suspension of strain YS05 diluted 100 times to 0.8) was drenched.

Treatment group 2: OD mixing with sterile Water₆₀₀Bacterial suspension of 0.8 strain YS21 was diluted 100 times, sprayed on the plants, and 50mL of treatment solution (OD) was added per plant until the leaves were covered with the solution without dripping water₆₀₀Bacterial suspension of strain YS21 diluted 100 times to 0.8) was drenched.

Treatment group 3: the 50% iprodione wettable powder (Somei plant protection agent Co., Suzhou, Jiangsu, registered number: PD20151441) was used in accordance with the instruction of the manufacturing company.

Control group: and (4) distilled water.

The spray inoculation concentration is 1 × 10 after 24h of treatment⁸cfu/mL of the tomato stolonifera fungus suspension is added to each treated plant, moisture is kept at 26-28 ℃, and the disease condition is investigated 10 days after treatment, wherein each treatment is 8 plants and is repeated for 3 times. Disease classification is carried out according to pesticide field efficacy test criteria (pesticide laboratory of Ministry of agriculture, 1993), and disease index and prevention and treatment effect are calculated.

The disease index ∑ (number of diseased leaves at each stage × number of disease stages)/(number of investigated total leaves × number of highest disease stages) × 100. Classification criteria of disease grade: level 0: no disease spots; level 1: the lesion area accounts for less than 5% of the whole leaf area; and 2, stage: the lesion area accounts for 6 to 25 percent of the whole leaf area; and 3, level: the lesion area accounts for 26-50% of the whole leaf area; 4, level: the lesion area accounts for 51 to 75 percent of the whole leaf area; and 5, stage: the lesion area accounts for more than 75% of the whole leaf area.

The preventing and treating effect (%) is (contrast disease index-treatment disease index)/contrast disease index x 100.

The results are shown in table 8 and fig. 8, and show that: the prevention and treatment effect of the strain YS05 on the leaf spot of the phoma stolonifera of the tomato reaches 61.27 percent, which is obviously higher than that of the control treatment of the medicament and the strain.

TABLE 8 determination of the control of the tomato stemphylium stolonifera potted plant by the strain YS05

Example 6 determination of Effect of strains YS05 and 6 drugs on prevention of in vitro leaves of Pholiota lycopersici

The in vitro leaf control effect of the strain YS05 and other 6 medicaments on the tomato stemphylium stolonifera is determined by a patch method, and the determination steps can be referred to a method in a document of Li B, Yuan H, Fang J, et al. The method comprises the following specific steps:

and (3) beating a fungus cake on the cultured tomato stemphylium stolonifera flat plate by using a 5mm sterile puncher, placing the tomato stemphylium stolonifera flat plate on the front side of the tomato leaves, and culturing for 3 days under the condition of light and dark alternation at the temperature of 28 ℃. Selecting strain YS05, inoculating single colony in KB liquid culture medium, shake culturing at 28 deg.C and 180rpm/min for 24 hr, and adjusting bacterial suspension concentration to OD₆₀₀Diluting the solution by 100 times, spraying and inoculating the diluted solution on tomato leaves containing a tomato stemphylium cake until the leaves are full of the solution and do not drip water. Distilled water was used as a blank (CK).

The YS05 bacterial suspension is respectively replaced by the following control agents for treatment: 8 hundred million/g waxy bacillus wettable powder (Shandong Tainuo pharmaceutical Co., Ltd., registration number: PD20094534)100 times liquid, 80 hundred million live spores/ml bacillus licheniformis water aqua (Guangxi jin Yan pesticide Co., Ltd., registration number: PD20140122)90 times liquid, 46 percent copper hydroxide water dispersible granule (U.S. DuPont, registration number: PD20110053)1800 times liquid, 33.5 percent quinoline copper suspending agent (Shandong ao Sheng biological science Co., Ltd., registration number: PD20200743)1500 times liquid, 10 percent polyoxin wettable powder (Shandong Texas Xianglong biochemical Co., Ltd., registration number: PD20100857)1000 times liquid, and 50 percent isoperide urea wettable powder (Jiangsu Suzhou Fumei plant protectant Co., registration number: PD20151441)800 times liquid.

Disease was investigated 7d after treatment, 6 leaves per treatment, 3 replicates. Calculating disease index and preventing and treating effect.

The disease index is 100 × Σ (number of diseased leaves at each stage × representative value of relative stage)/(total number of leaves × representative value of highest stage). Classification criteria of disease grade: level 0: no disease spots; level 1: the lesion area accounts for less than 5% of the whole leaf area; and 2, stage: the lesion area accounts for 6 to 25 percent of the whole leaf area; and 3, level: the lesion area accounts for 26-50% of the whole leaf area; 4, level: the lesion area accounts for 51 to 75 percent of the whole leaf area; and 5, stage: the lesion area accounts for more than 75% of the whole leaf area.

The control effect (%) is 100 x (control disease index-treatment disease index)/control disease index.

The results are shown in table 9 and fig. 9, and show that: the prevention effect of the bacterial suspension of the strain YS05 on the tomato gray leaf spot is 71.52 percent, which is obviously higher than that of other medicaments.

TABLE 9 determination of Effect of Strain YS05 and 6 drugs on in vitro leaf prevention of tomato phomopsis stolonifera

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> vegetable and flower institute of Chinese academy of agricultural sciences

<120> Pseudomonas chlororaphis and application thereof in prevention and treatment of leaf spot disease of phoma stolonifera

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1538

<212> DNA

<213> Artificial Sequence

<400> 1

ttaaggaggt gatccagccg caggttcccc tacggctacc ttgttacgac ttcaccccag 60

tcatgaatca caccgtggta accgtcctcc cgaaggttag actagctact tctggtgcaa 120

cccactccca tggtgtgacg ggcggtgtgt acaaggcccg ggaacgtatt caccgcgaca 180

ttctgattcg cgattactag cgattccgac ttcacgcagt cgagttgcag actgcgatcc 240

ggactacgat cggttttatg ggattagctc cacctcgcgg cttggcaacc ctctgtaccg 300

accattgtag cacgtgtgta gcccaggccg taagggccat gatgacttga cgtcatcccc 360

accttcctcc ggtttgtcac cggcagtctc cttagagtgc ccaccataac gtgctggtaa 420

ctaaggacaa gggttgcgct cgttacggga cttaacccaa catctcacga cacgagctga 480

cgacagccat gcagcacctg tctcaatgtt cccgaaggca ccaatccatc tctggaaagt 540

tcattggatg tcaaggcctg gtaaggttct tcgcgttgct tcgaattaaa ccacatgctc 600

caccgcttgt gcgggccccc gtcaattcat ttgagtttta accttgcggc cgtactcccc 660

aggcggtcaa cttaatgcgt tagctgcgcc actaagagct caaggctccc aacggctagt 720

tgacatcgtt tacggcgtgg actaccaggg tatctaatcc tgtttgctcc ccacgctttc 780

gcacctcagt gtcagtatca gtccaggtgg tcgccttcgc cactggtgtt ccttcctata 840

tctacgcatt tcaccgctac acaggaaatt ccaccaccct ctaccatact ctagctcgcc 900

agttttggat gcagttccca ggttgagccc ggggatttca catccaactt aacgaaccac 960

ctacgcgcgc tttacgccca gtaattccga ttaacgcttg caccctctgt attaccgcgg 1020

ctgctggcac agagttagcc ggtgcttatt ctgtcggtaa cgtcaaaata ctcacgtatt 1080

aggtaagtac ccttcctccc aacttaaagt gctttacaat ccgaagacct tcttcacaca 1140

cgcggcatgg ctggatcagg ctttcgccca ttgtccaata ttccccactg ctgcctcccg 1200

taggagtctg gaccgtgtct cagttccagt gtgactgatc atcctctcag accagttacg 1260

gatcgtcgcc ttggtgagcc attacctcac caactagcta atccgaccta ggctcatctg 1320

atagcgcaag gcccgaaggt cccctgcttt ctcccgtagg acgtatgcgg tattagcgtc 1380

cgtttccgga cgttatcccc cactaccagg cagattccta ggcattactc acccgtccgc 1440

cgctctcaag agaagcaagc ttctctctac cgctcgactt gcatgtgtta ggcctgccgc 1500

cagcgttcaa tctgagccat gatcaaactc ttcagttc 1538

<210> 2

<211> 2418

<212> DNA

<213> Artificial Sequence

<400> 2

tcagaagtcc aggttggata ccgccagggc gttgctttcg atgaagtcgc gacgaggctc 60

gaccgcatcc cccatcaggg tgttgaagat ctggtcggcg ccgatggcgt cttcgatggt 120

gaccttgagc atacggcgca cgcttgggtc catggtggtt tcccacagct gatccgggtt 180

catctcgccc agacctttgt atcgctggat ggtgtggcgc ttggtgcttt cggtcatcag 240

ccagtccagg gcttccttga actcggtcac cgccttcttg cgttcgccgc gctgaatata 300

agcaccttcg tccagcaggg agctcagttg agcgccgagg gtaacgacgg tcttgtagtc 360

attgctgccg aagaagtcgc ggttgaaggt gacgtagttc gacaggccgt gggagatcag 420

ttcgacctct ggcagccaga cattacgttc acggtcttca cgcaggctgg ctttgtaaac 480

caggccggac ttctcgacgg tgcgcaggcg gacttcatat tgggccagcc aatcctgcat 540

cgccgcgtga tcggagagtt gctccaggct cacggccggc aggtagatga agtgctcggt 600

cagctcctga gggtacagcc gcgacagacg cttgagggtt ttcatgacca tgcggaagtc 660

gttcaccagg cgctccagcg cctcgccgga aatacccggt gcgtcttcgt tcaggtgcag 720

gctcgcatct tccagggccg actgcgtcat gtactcttcc atggcatcgt cgtctttgat 780

gtattgctct tgcttgcctt tcttgacctt gtacagcggc ggctgagcga tatagatgta 840

gccacgctcg atcagctccg gcagctgacg gaagaagaag gtcagcagca gggtacggat 900

gtgcgaaccg tcgacgtcgg cgtcggtcat gatgatgatg ttgtgataac gcagcttgtc 960

gatgttgtac tcttcgcggc cgatgccgca accgagtgca gtgatcaagg tgccgacctc 1020

ttgcgaggaa atcatcttgt cgaagcgcgc tttctcgacg ttaaggatct tgcccttgag 1080

tggcagaatc gcctgggtct tacggttgcg tccctgcttg gcggagccgc cagcagagtc 1140

accttccacc aggtagagtt cggaaagggc agggtctttc tcctggcagt ccgccagttt 1200

gcccggcagg ccggcgatat ccagcgcgcc tttacggcgg gtcatctcgc gagccttacg 1260

cgccgcttca cgggcacggg cggcgtcgag catcttgccg accaccagct tggcttcgtt 1320

cgggttttcc agcaggaagt cggagaagta cctgcccatt tcctgttcga ccgcggtctt 1380

cacttcggaa gacaccagct tgtctttggt ctgggagctg aacttcggat ccggcacctt 1440

gaccgaaatg atcgcggtca ggccttcacg ggcatcgtca ccggtggtgg cgaccttgtg 1500

cttcttcgcc agaccttcct gttcgatgta attgttcagg ttacgcgtca gtgccgaacg 1560

gaagcccacc aggtgggtgc cgccgtcgcg ctgcggaatg ttgttggtga agcactgcag 1620

gttttcgttg aagctgtcgt tccactgcag ggcgatttcc acgccgatgc catcttcacg 1680

ttgcacattg aagtggaaca cctggttgac cgcggtcttg ttggtgttca ggtattcaac 1740

gaatgcacgc aggccgcctt cgtacttgaa cagttcttcc ttgccgctgc gttcgtcctt 1800

cagaacgata ccgacaccgg agttgaggaa ggacagttca cgaatccgct tggccaggat 1860

gtcccagctg aaatggatgt tcttgaaggt ctcgctggaa gccttgaagt gaatctgggt 1920

accggtggtt tcgctgtcac cgacgatcgc cataggcgcc tgaggtacac cgtgaacgta 1980

ggtctgctcc cagatcttgc cgctgcggcg aacggtcagg accagttctt cggacaaggc 2040

gttcactacc gacacaccca caccgtgcag accgccggat actttgtagg agttgtcgtc 2100

gaacttaccg ccggcgtgca gcacggtcat gatgacctcg gccgcggaaa cgccttcttc 2160

tttatgcacg tctaccggga tgccgcgacc gttgtcacgc acggtaatgg attcgtccgg 2220

atggatgatg atgctgatgt cgtcgcagtg gccggccaga gcttcgtcga tcgagttatc 2280

gaccacctcg aacaccatat ggtgcagacc gctgccatca tcggtgtcac caatgtacat 2340

accgggacgt ttgcgtacgg catccaaacc tttcagcact ttaatgctgc tcgagtcgta 2400

cgtgttttct tcgctcat 2418

<210> 3

<211> 4074

<212> DNA

<213> Artificial Sequence

<400> 3

atggcttact catatactga gaaaaaacgt atccgcaagg actttagcaa gttgccggac 60

gtcatggatg tgccgtacct cctggccatc cagctggatt cgtatcgtga attcttgcaa 120

gcgggagcga ctaaagatca gttccgcgac gtgggcctgc atgcggcctt caaatccgtt 180

ttcccgatca tcagctactc cggcaatgct gcgttggagt atgtcggtta tcgcctgggc 240

gaaccggcat ttgatgtcaa agaatgcgtg ctgcgcggtg taacttacgc cgtacctttg 300

cgggtaaaag ttcgtctgat cattttcgac aaagagtcgt cgaacaaagc gatcaaggac 360

atcaaagagc aagaagtcta catgggtgaa atccccctga tgactgagaa cggtaccttc 420

gtaatcaacg gtaccgagcg tgtaatcgtt tcccagctgc accgttcccc tggcgtattc 480

ttcgaccacg accgtggcaa gacgcacagc tccggtaagc tgctgtactc cgcacgtatc 540

attccttacc gcggttcctg gttggacttc gagttcgacc cgaaagactg cgtattcgtg 600

cgtatcgacc gtcgccgcaa gctgcctgca tcggtgctgc tgcgtgcgct gggctacacc 660

actgaagaag tgctggacgc tttctacacc accaacgtat tccacgtgcg cggcgaaagc 720

ctgagcctgg aactggtgcc tcagcgcctg cgtggtgaaa ttgccgtcct cgatattcag 780

gatgacaaag gcaaggttat tgtcgagcag ggtcgtcgta ttaccgctcg ccacatcaac 840

cagctggaaa aagccgggat caaagagctg gaagtgccgc tggactatgt cctgggtcgc 900

actaccgcca aggtcatcgt gcatccggcc accggcgaaa tcctggcaga gtgcaacacc 960

gagctgaaca ccgagatcct ggcgaagatc gccaaggctc aggttgtgcg catcgaaacg 1020

ctgtacacca acgatatcga ttgcggtccg ttcgtttccg acactctgaa gatcgactcc 1080

accaccaacc agctggaagc gttggtcgag atctatcgca tgatgcgtcc tggcgagcct 1140

ccaaccaagg atgcagccga gaccctgttc aacaacctgt tcttcagccc tgagcgctat 1200

gacctgtctg cggtcggccg gatgaagttc aaccgtcgta tcggccgtac cgagatcgaa 1260

ggttcgggcg tgttgtgcaa ggaagacatc gttgcggttc tgaagaccct ggtcgacatc 1320

cgtaacggca aaggcatcgt cgatgacatc gaccacctgg gtaaccgtcg tgttcgctgc 1380

gtaggcgaga tggccgagaa ccagttccgc gttggcctgg tgcgcgtaga gcgtgcggtc 1440

aaagagcgtc tgtcgatggc tgaaagcgaa ggcctgatgc cgcaagacct gatcaacgcc 1500

aagccagtgg ctgcggcggt gaaggagttc ttcggttcca gccagctgtc ccagttcatg 1560

gaccagaaca acccgctgtc cgagatcacc cacaagcgtc gtgtctctgc cctcggccca 1620

ggcggtctga ctcgtgagcg tgcaggcttc gaagtgcgtg acgtacaccc gactcactat 1680

ggtcgtgtat gtccgattga aacgccggaa ggtccgaaca tcggtctgat caactccttg 1740

gctgcctatg ctcgcaccaa tcagtatggc ttcctcgaga gcccgtaccg cgtggtgaaa 1800

gaaggtctgg tgaccgacga gatcgtgttc ctgtccgcta tcgaagaggc cgatcacgtg 1860

atcgcccagg cttcggcgac catgaacgac aaaggtcagc tgatcgacga gctggtagct 1920

gttcgtcacc tgaacgaatt caccgtcaag gcgccagaag acgtcacctt gatggacgtt 1980

tcgccgaagc aggtagtttc ggttgcggcg tcgctgattc cgttcctcga gcacgacgac 2040

gccaaccgtg cgttgatggg ttcgaacatg cagcgtcagg ctgtaccaac cctgcgtgcc 2100

gacaagccgc tggtaggtac cggcatggag cgtaacgttg cccgtgactc cggcgtctgc 2160

gtcgtggctc gtcgtggcgg cgtgatcgac tcggttgatg ccagccgtat cgtggttcgt 2220

gttgcggatg acgaagttga accaggcgaa gccggtgtcg acatctacaa cctgaccaaa 2280

tacacccgct ccaaccagaa cacctgcatc aaccagcgtc cgctggtgag caagggtgat 2340

cgggttcagc gcagcgatat catggccgac ggtccgtcca ccgatatggg tgaactggct 2400

ctgggtcaga acatgcgcat cgcgttcatg gcatggaacg gcttcaactt cgaagactcc 2460

atctgcctgt ccgagcgtgt ggttcaggaa gaccgcttca ccacgatcca catccaggaa 2520

ctgacctgtg tggcccgtga caccaagctt ggcccagagg aaatcactgc ggacatcccg 2580

aacgtgggtg aggctgcact gaacaagctg gacgaagccg gtatcgttta tgtaggtgct 2640

gaagttggtg caggcgacat cctggtcggt aaggtcactc cgaaaggcga gacccagctg 2700

actccggaag aaaaactgct gcgtgcaatc ttcggtgaaa aagccagcga cgttaaagac 2760

acctccctgc gcgtacctac cggtaccaag ggtactgtca tcgacgtaca ggtcttcact 2820

cgtgacggcg tagagcgtga cgctcgtgcc ctgtcgatcg agaagagcca gctcgacgag 2880

atccgcaagg atctgaacga agagttccgt atcgtcgaag gcgccacttt cgaacgtctg 2940

cgttccgctc tggtcggcca caaagccgaa ggcggcgccg gtctgaagaa ggggcaggaa 3000

atcaccgacg aagtactcga cggtcttgag catggtcagt ggttcaaact gcgcatggct 3060

gaagatgctc tgaacgagca gcttgagaag gcccaggcct atatcgttga tcgccgccgt 3120

ctgctggacg acaagttcga agacaagaag cgcaaactgc agcagggcga tgacctggcc 3180

ccaggcgtgc tgaaaatcgt caaggtttac ctggcaatcc gtcgtcgcat ccagccgggc 3240

gacaagatgg ccggtcgtca cggtaacaag ggtgtggtct ccgtgatcat gccggttgaa 3300

gacatgccgc acgatgccaa tggcaccccg gtcgatgtcg tcctcaaccc gttgggcgta 3360

ccttcgcgta tgaacgttgg tcagattctc gaaacccacc tgggcctcgc ggccaagggg 3420

ctgggcgaga agatcaaccg catggtcgaa gagcagcgca aggtcgcgga actgcgtaaa 3480

ttcctggacg agatctacaa ccagatcggt ggtcgtaacg aagatctgga tagcttctcc 3540

gatcaggaaa tcctggatct ggcgaaaaac ctgcgtggcg gcgtgccgat ggccactccg 3600

gtgttcgacg gtgccaagga aagcgaaatc aaggccatgc tgaaactggc ggacctgcca 3660

gaaagcggcc agatgcagct gaccgacggc cgtaccggca acaagttcga gcgcccggtt 3720

actgttggct acatgtacat gctgaagctg aaccacttgg tagacgacaa gatgcacgct 3780

cgttctaccg gttcgtacag cctggttacc cagcagccgc tgggtggtaa ggcgcagttc 3840

ggtggtcagc gtttcgggga gatggaggtc tgggcactgg aagcatacgg tgctgcatac 3900

actctgcaag aaatgctcac agtgaagtcg gacgatgtga acggccggac caagatgtac 3960

aaaaacatcg tggacggcga tcaccgtatg gagccgggca tgcccgagtc cttcaacgtg 4020

ttgatcaagg aaattcgttc cctcggcatc gatatcgatc tggaaaccga ataa 4074

<210> 4

<211> 1848

<212> DNA

<213> Artificial Sequence

<400> 4

ttactcgtcg aggaaggagc gcaggtgctc gcttcgcgtc ggatggcgca gcttgcgcag 60

cgccttggct tcgatctggc gaatccgctc acgggtaacg tcgaactgct taccgacttc 120

ctcgagggtg tggtcggtat tcatgtcgat gccgaagcgc atgcgcagta ccttggcttc 180

acgggcagtg aggccggaga gtacttcgcg agtcgcttct ttaaggctct cgacggtggc 240

gacatcgatt ggcgactgca tggtcgagtc ttcgatgaag tcacccagat gggagtcttc 300

gtcatcaccg atcggggttt ccatggagat cggctcttta gcgatcttca ataccttgcg 360

gatcttgtcc tcaggcattt ccatgcgttc gcccagctct tccggagtcg gttcgcgacc 420

catttcctgc aacatctgcc gggaaatacg gttgagcttg ttgatcgtct cgatcatgtg 480

caccggaata cgaatggtgc gggcctggtc ggcgatcgag cgagtgatcg cctgacggat 540

ccaccaggtg gcataagtcg agaacttgta gccgcgacgg tattcgaact tgtctaccgc 600

tttcatcaaa ccgatgttgc cttcctggat caggtcgagg aattgcaggc cacggttggt 660

gtacttcttg gcgatggaga tcaccaggcg caggttggct tcgaccatct ctttcttcgc 720

gcgacgggcc ttggcctcgc cgatcgacat gcgacggttg atgtccttga tctcggcgat 780

cgtcaggccg gtctcggtct cgagcgcggt cagcttctgc tggcaacgga tgatgtccgg 840

ctgcaggcgg ccgatggctt cggcgtactt ggccttgcct ttggccagtg cgtcggacca 900

gctttcgtcc acttcattgc cagggaactg gcgcaggaag tcggcgcgtg gcatgcgggc 960

atcacgaaca cagagctgca tgatcgcgcg ctcttgctga cgcaggcgat ccagggcgct 1020

acgaacacgt tcaaccaagc cttcgaattg cttcggaacc agtttgatcg gcatgaacag 1080

ttcagccagg gccagcattt cagccagggc ttgcttgtgt tcgcgaccgt gcttcttcag 1140

cgccttgcgg gtgatttcca tctggtcggc aacggcgcca aagcgctggg ctgcgatgac 1200

cggatccgga ccgctttcgg cttcttcttc gtcgtcactg gcttcggcgt catcgtcgtc 1260

ggtgtcgtcg tccgcttttg tggccttggc atcgacaggc ggtggtactt cggcggcagg 1320

cggcgcaatg ccgtcgtccg ggtcgatgta accgctcagg acgtcggaca ggcggccacc 1380

ttcggtggtg acgcgagtgt attcggagag gatgtgctca accgtgccag ggaagtgcgc 1440

gatcgcgccc atcacctcac ggatgccctc ttcgatacgc ttggcgattt cgatttcgcc 1500

ttcacgtgtg agcagctcta ccgtacccat ttcgcgcatg tacatacgca ctgggtcggt 1560

agtgcgacca atgtcggttt ccacagccgc caacgctgcg gcggcttctt cagctgcagc 1620

ttcgtcagta tcggcttcgg ccaacaaaag ggcatccgca tccggagcac tctcgaatac 1680

gttgatcccc atgtcgttga tcatgcggat gatgtcttcc acctgttccg gatctgaaat 1740

atcctccggc aggtggtcgt tgacctccgc gtaagtcagg taaccctgct cacgaccacg 1800

gctgatcaac tctttgaggc gagactgctg ttgcgctttt ccggacat 1848

Claims

1. Pseudomonas chlororaphis subsp.

2. The bacterial suspension or culture solution or fermentation broth or fermentation product or volatile substance of Pseudomonas chlororaphis subsp.

3. Use of a bacterial suspension or culture broth or fermentation product or volatile substance of Pseudomonas chlororaphis subsp.

4. Use of a bacterial suspension or culture broth or fermentation product or volatile substance of Pseudomonas chlororaphis subsp.

5. Use of a bacterial suspension or culture solution or fermentation broth or fermentation product or volatile substance of Pseudomonas chlororaphis subsp.

6. Use of a bacterial suspension or culture solution or fermentation broth or fermentation product or volatile substance of Pseudomonas chlororaphis subsp.

7. Use of a bacterial suspension or culture broth or fermentation product or volatile substance of Pseudomonas chlororaphis subsp.

8. Use of a bacterial suspension or culture broth or fermentation product or volatile substance of Pseudomonas chlororaphis subsp.

9. A product, wherein the active ingredient is a bacterial suspension or culture solution or fermentation broth or fermentation product or volatile substance of the pseudomonad chlororaphis subsp.

The product has any one of the following functions of a1) -a 3):

a1) inhibiting pathogenic bacteria;

a2) preventing and treating plant diseases caused by pathogenic bacteria;

a3) preventing and treating the leaf spot of the botrytis cinerea.

10. Any one of the following b1) -b 3):

b1) a method of suppressing a pathogenic bacteria comprising the steps of: treating a pathogenic bacterium with a bacterial suspension or culture solution or fermentation broth or fermentation product or volatile substance of Pseudomonas chlororaphis subsp.

b2) A method of controlling plant diseases caused by pathogenic bacteria, comprising the steps of: treating a plant with a bacterial suspension or culture broth or fermentation product or volatile substance of Pseudomonas chlororaphis subsp.

b3) A method for preventing and treating a leaf spot disease of tomato stemphylium stolonifera comprises the following steps: treating tomatoes with a bacterial suspension or culture broth or fermentation product or volatile substance of Pseudomonas chlororaphis subsp.